Summary: Impaired wound healing following injury in diabetics represents a major clinical problem, resulting in prolonged hospitalizations and significant healthcare costs. Two-thirds of all non-traumatic amputations are preceded by a diabetic wound. The development of diabetic peripheral neuropathy increases the susceptibility of diabetic skin to injury. We have shown that diabetic skin in mice and humans also has impaired skin integrity at baseline which further predisposes diabetic skin to injury. Diabetic wounds are deficient in stromal derived factor-1??(SDF-1?), a potent chemokine involved in progenitor cell recruitment, angiogenesis, and granulation tissue formation, mediated through binding to the CXCR4 receptor and the establishment of a chemotactic gradient. We have shown overexpression of SDF-1? corrects the diabetic wound healing impairment, and in exciting preliminary studies, can restore the integrity of diabetic skin. Given this important clinical problem, the objective of this proposal is to develop a small molecule therapeutic to target the SDF-1? receptor CXCR4 to improve diabetic skin integrity to prevent injury, and to improve wound healing should injury occur. We have developed and optimized an innovative screening approach to identify new classes of selective CXCR4 receptor agonists that can be applied topically and penetrate the skin to improve skin integrity and wound healing. We propose to screen the NIH SMR library and carry out hit-to-lead studies to identify novel CXCR4 receptor agonists that can be developed for these complications of diabetes. Aim 1. We will screen the NIH compound collection to identify molecules that can agonize the cAMP signaling pathway via the CXCR4 receptor. We have developed and optimized the primary assay to screen for first in class CXCR4 small molecule agonists and expanded our pilot screen to include 6400 compounds from our internal collection to justify screening the entire NIH SMR library using our robust testing funnel. Aim 2. We will implement dose response studies, validate all ?Hits? using a counter-screen and secondary chemotaxis/migration assays, confirm direct receptor binding, and optimize our most promising molecules using SAR by catalog and medicinal chemistry approaches. Hits from Aim 1 will be counter-screened for selectivity against the related CXCR5, CCR6, CXCR6 receptors and unrelated APJ receptors. Functional assays using human cells will assess chemotaxis and migration. Cell-based binding studies will confirm direct binding to receptor, binding parameters, and optimize promising candidates by SAR. Aim 3. We will examine the ability of validated target molecules from Aim 2 to correct the abnormal expression of microRNAs that regulate inflammation, angiogenesis, and collagen synthesis in human diabetic fibroblasts, and whose expression is corrected by SDF-1?. Target molecules from Aim 2 will be screened for their ability to correct expression of microRNA-146a, 15b, and 29a, which are dysregulated in diabetes, and corrected with SDF-1? treatment.